Rickettsia prowazekii is one of the most lethal bacterial pathogens known to man; it has decimated entire armies and has been weaponized. It is a category B biothreat select agent because of transmissibility by aerosol, low infectious dose, high stability, and causation of severe illness with a high case-fatality ratio. The development of a cross-reactive vaccine is feasible because natural rickettsial infection provides protective immunity in survivors of epidemic (as well as murine) typhus, and because cross-protection has been demonstrated experimentally; nevertheless, safe and effective vaccines do not exist because of our incomplete understanding of the immune correlates of protection, and because the protective antigens remain unidentified. Thus, the objective of this application is to discover cross-protective rickettsial antigens recognized by T and B lymphocytes. We hypothesize that a subunit vaccine based on cross-reactive rickettsial antigens will protect mice, and a novel humanized mouse model, from lethal homologous and heterologous rickettsial challenges. This investigation will contribute to the long-term goal of developing immunotherapeutic agents for humans against rickettsiae. Five specific aims with genome-wide approaches will address the hypothesis: 1) identify immunological correlates of protection against rickettsial infection; 2) identify new MHC class l-restricted cross-reactive rickettsial antigens recognized by CD8+ T lymphocytes; 3) identify MHC class ll-restricted cross-reactive rickettsial antigens recognized by CD4+ T lymphocytes; 4) identify cross-reactive rickettsial antigens recognized by anti-rickettsial antibodies; and 5) determine the protection conferred by a subunit vaccine against a lethal challenge with Rickettsia. The first aim will provide critical definitions of immune correlates of protection that will guide the selection and evaluation of vaccination protocols. Aims 2, 3, and 4 will identify rickettsial T cell and B cell antigens through a systematic evaluation of all R. prowazekii genes. The fifth aim will identify a vaccine formulation and vaccination protocol that will provide a durable multifunctional and cross-reactive protective immune response. In this way, our research directly addresses WRCE's strategic plan and NIAID's Biodefense Research Agenda. The innovation of this research lies in the systematic definition of immune correlates of protection, the discovery of rickettsial antigens through a comprehensive genomic screen, the testing of multiple vaccine formulations, and the use of a highly relevant mouse model with a reconstituted functional human immune system.